Research Area:
Muscle Tissue Engineering

Robert G. Dennis, Ph.D.

Bob's Home Page   Current Research    Muscle Mechanics Lab (U of M)     Biomechatronics Group @ MIT

Objective:  To engineer functional contractile mammalian skeletal muscle in vitro.

[view movie of spontaneously contracting engineered muscle]

    The potential applications for functional engineered skeletal muscle extend from basic research to drug discovery to surgical transplantation, hybrid prosthetics, and robotics, and perhaps on even to include engineered and farmed animal protein as a food source.  The adult human body is approximately 40% skeletal muscle by mass.  Diseases of skeletal muscle range from debilitating to crippling and lethal.  The ability to take a few cells from an adult mammal (or human) and produce a large mass of functional skeletal muscle would be of incalculable benefit to man kind.

    The State of the Art in Functional Skeletal Muscle Tissue Engineering can be easily summarized by first defining the function of skeletal muscle.  Though muscle tissue performs many functions for the body, some arising from the emergent properties of muscle cells organized into whole muscle organs, such as heat generation and protein synthesis, the most basic definition of muscle tissue function is the generation of controlled force, work, and power.  It is necessary to quantify the contractility of the muscle tissue, to organize the tissue in such a way as to promote the generation of directed force, and exert control over the contractions for research in this area to be considered engineering, rather than cell biology.  After all, spontaneous contractions in cultured skeletal muscle cells were first reported in 1915 (Lewis), and this was not construed as 'engineering'.  Defining Functional Skeletal Muscle Tissue Engineering in this way, it is possible to assert that at this time there are only three research groups in the world engineering functional skeletal muscle in vitro: Herman Vandenburgh and Paul Kosnik in Providence, RI; myself and Hugh Herr at MIT, and the Muscle Mechanics Laboratory at the University of Michigan.

Experimental Approach:
    My principle collaborators in this effort are Paul Kosnik, Ph.D. and Herman Vandenburgh, Ph.D.  Herman has a lengthy history of functional skeletal muscle tissue engineering, his first papers in the area being published in the 1980's.  My collaboration with Hugh Herr is focused on robotic actuator technologies involving engineered muscle, and includes muscle tissue from many sources other than mammalian.
    The experimental approach developed by Paul and I centered on developing self organizing muscle tissues, that is, employing no artificial scaffolds for the contractile segment of the engineered muscle.  In 1997 and 1998 we developed a baseline model for engineered muscle in vitro that was self organizing, and generated easily measurable force.  We term these engineered skeletal muscle constructs myooids, because they are muscle like in form and function.  The self organization of myooids in culture is shown below.  Note that the muscle remains attached at each end to a silk suture anchor.  The suture anchors serve as artificial tendons, allowing the muscle to be manipulated and attached to force transducers and servo motors without injuring the muscle.

The image to the left is a photograph of the first myooid ever produced in our laboratory (July 7, 1997).  The myooid is attached at each end to acellularized skeletal muscle anchors, pinned in place by 0.10" diameter insect pins.  This myooid is approximately 30 mm in length, from anchor to anchor.  It was cultured in a 100 mm diameter culture dish, with an uncoated Sylgard substrate.

The myooid was seen to spontaneously contract immediately after each feeding.  No functional data were measured, as the instrumentation was not in place at that time.

(Photograph by R.G. Dennis and P. Kosnik, 1997)

Myooid self organizing by cell monolayer delamination
Fully formed myooid, ~3 days later
X-section of a smaller diameter myooid
Cross section of a larger diameter myooid
1. Monolayer of muscle cells detaching from the substrate and rolling into a cylinder.  This process requires several days.  Dish diameter is 35 mm. 2.  Approximately 3 days after delamination of the monolayer, the cells have self organized into a cylinder. 3.  Representative cross section of a myooid, stained with 1% Toluidine blue.  The scale bar is 100 mm.  Note the annulus of fibroblasts surrounding the muscle cells. 4.  Representative cross section of a myooid, stained with 1% Toluidine blue.  The scale bar is 100 mm.

Functional Data:
    We have measured the contractility and excitability of over 1500 myooids since 1998.  The process for making myooids is highly reliable (98%) and scalable.  It is possible to make 500 to 1000 myooids from the muscle tissue of a single adult rat.  Each myooid can be thoroughly characterized to quantify the excitability and contractility.  Representative data is given below.
Spontaneous contractions of a myooid, ~1 Hz. A single twitch, elicited by a single, 70V, 0.4 ms pulse. Tetanic contractions at 30 Hz (black trace) and 6 Hz (red trace). Force frequency diagram.  As stimulation frequency is increased, tetanic force increases.

Length-tension curve.  Note that the passive curve (black) is left shifted from control values.  The peak of the active curve (red) occurs at -5% to -10% of the cultured length. Effect of caffeine addition on the contractile force of myooids.  this demonstrates that myooids could be used in a contractility assay to diagnose malignant hyperthermia. Diffusion limits the size of avascular engineered skeletal muscle.  The maximum diameter is generally 500 mm.  Myooids with diameters greater than this have necrotic cores, with essentially non-contractile muscle.

Internal forces generated within a myooid:
Myooids are a co-culture of at least two cell types; myoblasts (which form long tubes) and fibroblasts.  During self organization, the fibroblasts are essential for forming the extracellular matrix material that holds the structure together.  After formation, fibroblasts tend to organize around the periphery of the myooids, as shown to the left.  Based on our passive force data, and using a multiple regression model (assuming biaxial tension generation by the fibroblasts), we calculate that the fibroblasts exert a continuous 5 kPa stress, which acts both longitudinally and as a hoop stress.  The hoop stress results in an internal pressure of ~ 1 kPa on the myotubes at the core of the myooid.
Myooid from adult muscle, 
~ 7% fibroblasts in the 
cross section.
Myooid from neonatal 
cells, ~ 30% fibroblasts 
in the cross section.

Tissue excitability:
    Tissue excitability is an extremely important measure of function.  When stimulating electrically, the excitability will define the power requirements to depolarize the muscle cell membranes to elicit a contraction.  For chronic stimulation in a bioreactor, or for use in a robotic or prosthetic application, the tissue excitability will be the dominant term when calculating the electrical power consumption during electrical stimulation of the tissue.
Bulk excitability of an engineered muscle
Rheobase (R50):  Stimulus amplitude required to elicit a 50% peak twitch force. (measured at long stimulus pulse duration)
Bulk excitability of an engineered muscle
Chronaxie (C50):  Stimulus duration required to elicit a 50% peak twitch force.
(measured at 2x R50)
Classical definition of excitability for nerve and muscle tissues.  The combination of stimulus duration (chronaxie) and stimulus strength (rheobase) must lie above the curve to elicit depolarization of the cell membrane.

Based on our measurements of the excitability of engineered tissues, the values for R50 and C50 are each approximately 1 order of magnitude greater than control muscle tissues.  High values of C50 and R50 indicate LOW tissue excitability, which is undesirable.  This is understandable, given that the engineered tissues are cultured entirely in the absence of innervation.  The consequence of this very low excitability is that the energy required to stimulate the tissues is much greater than required for normally innervated muscle; it is in fact ~1000 times greater, calculating the energy requirements directly from Ohm's law.  Thus, it is critical to improve the excitability of engineered skeletal muscle tissue if the tissue is ever to be exposed to chronic electrical stimulation in culture, or if it is to function as a robotic or prosthetic actuator.

Movie of the Spontaneous Contractions of a Myooid

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Spontaneously contracting myooid with wide insertion. Click on the image to the left to view a movie of a spontaneously contracting engineered muscle, a "myooid".  On the right, the myooid is attached to a silk suture anchor.  To the left, the myooid tapers to a diameter of approximately 400 microns.  The structure is opaque because if its thickness.  Myooids are generally cylindrical in cross section, ranging from 0.1 to 1.1 mm in diameter.  The thin vertical fiber at the top is just debris in the culture medium.

Discussion of Movie:  Note that in this case the myooid has naturally reduced the stress concentration at its attachment point by increasing its section thickness gradually as it inserts into the suture.  The muscle fibers interdigitate into the silk fibers of the suture.  The spontaneous contractions are vigorous and occur at a high frequency (several Hz) because the culture media surrounding this myooid has just been replaced.  In a short while, the spontaneous contractions will tend to slow to ~ 1 Hz.

Current and Future research:
    My future research will be directed toward integrating nerve and muscle tissue in vitro to increase the excitability of the tissue and enhance the expression of adult phenotypes, the application of mechanical and electrical interventions to promote growth and development of the tissue, improved mechanical interface between the muscle and its attachment points using tissue engineered tendons, and ultimately the addition of a vascular system with self-organizing vascular endothelium.

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